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- EMDB-50947: Xenopus tropicalis undecorated microtubule - 15 protofilament, 3-... -

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Basic information

Entry
Database: EMDB / ID: EMD-50947
TitleXenopus tropicalis undecorated microtubule - 15 protofilament, 3-start helix
Map data
Sample
  • Complex: Undecorated tubulin lattice following polymerisation with GTP (no stabilising ligands).
    • Protein or peptide: Tubulin beta chain
    • Protein or peptide: Tubulin alpha chain
  • Ligand: GUANOSINE-5'-DIPHOSPHATE
  • Ligand: GUANOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
KeywordsMicrotubule Tubulin Cytoskeleton Filament / PROTEIN FIBRIL
Function / homology
Function and homology information


microtubule-based process / structural constituent of cytoskeleton / microtubule / hydrolase activity / GTPase activity / GTP binding / cytoplasm
Similarity search - Function
Tubulin-beta mRNA autoregulation signal. / Alpha tubulin / Beta tubulin, autoregulation binding site / Beta tubulin / Tubulin / Tubulin, C-terminal / Tubulin C-terminal domain / Tubulin, conserved site / Tubulin subunits alpha, beta, and gamma signature. / Tubulin/FtsZ family, C-terminal domain ...Tubulin-beta mRNA autoregulation signal. / Alpha tubulin / Beta tubulin, autoregulation binding site / Beta tubulin / Tubulin / Tubulin, C-terminal / Tubulin C-terminal domain / Tubulin, conserved site / Tubulin subunits alpha, beta, and gamma signature. / Tubulin/FtsZ family, C-terminal domain / Tubulin/FtsZ-like, C-terminal domain / Tubulin/FtsZ, C-terminal / Tubulin/FtsZ, 2-layer sandwich domain / Tubulin/FtsZ family, GTPase domain / Tubulin/FtsZ family, GTPase domain / Tubulin/FtsZ, GTPase domain / Tubulin/FtsZ, GTPase domain superfamily
Similarity search - Domain/homology
Tubulin beta chain / Tubulin alpha chain
Similarity search - Component
Biological speciesXenopus tropicalis (tropical clawed frog)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsTroman LA / Moores CA
Funding support United Kingdom, 4 items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MR/R000352/1 United Kingdom
Medical Research Council (MRC, United Kingdom)MR/Y000633/1 United Kingdom
Wellcome Trust202679/Z/16/Z United Kingdom
Wellcome Trust206166/Z/17/Z United Kingdom
CitationJournal: Curr Biol / Year: 2025
Title: Mechanistic basis of temperature adaptation in microtubule dynamics across frog species.
Authors: Luca Troman / Ella de Gaulejac / Abin Biswas / Jennifer Stiens / Benno Kuropka / Carolyn A Moores / Simone Reber /
Abstract: Cellular processes are remarkably effective across diverse temperature ranges, even with highly conserved proteins. In the context of the microtubule cytoskeleton, which is critically involved in a ...Cellular processes are remarkably effective across diverse temperature ranges, even with highly conserved proteins. In the context of the microtubule cytoskeleton, which is critically involved in a wide range of cellular activities, this is particularly striking, as tubulin is one of the most conserved proteins while microtubule dynamic instability is highly temperature sensitive. Here, we leverage the diversity of natural tubulin variants from three closely related frog species that live at different temperatures. We determine the microtubule structure across all three species at between 3.0 and 3.6 Å resolution by cryo-electron microscopy and find small differences at the β-tubulin lateral interactions. Using in vitro reconstitution assays and quantitative biochemistry, we show that tubulin's free energy scales inversely with temperature. The observed weakening of lateral contacts and the low apparent activation energy for tubulin incorporation provide an explanation for the overall stability and higher growth rates of microtubules in cold-adapted frog species. This study thus broadens our conceptual framework for understanding microtubule dynamics and provides insights into how conserved cellular processes are tailored to different ecological niches.
History
DepositionJul 8, 2024-
Header (metadata) releaseJan 15, 2025-
Map releaseJan 15, 2025-
UpdateFeb 12, 2025-
Current statusFeb 12, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_50947.png

Downloads & links

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Map

FileDownload / File: emd_50947.map.gz / Format: CCP4 / Size: 274.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 416 pix.
= 443.872 Å		1.07 Å/pix.
x 416 pix.
= 443.872 Å		1.07 Å/pix.
x 416 pix.
= 443.872 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.067 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.015
Minimum - Maximum-0.023292147 - 0.06483061
Average (Standard dev.)0.00043404443 (±0.004322281)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions416416416
Spacing416416416
CellA=B=C: 443.872 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_50947_msk_1.map
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Additional map: #1

Fileemd_50947_additional_1.map
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Half map: #1

Fileemd_50947_half_map_1.map
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Half map: #2

Fileemd_50947_half_map_2.map
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Sample components

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Entire : Undecorated tubulin lattice following polymerisation with GTP (no...

EntireName: Undecorated tubulin lattice following polymerisation with GTP (no stabilising ligands).
Components
  • Complex: Undecorated tubulin lattice following polymerisation with GTP (no stabilising ligands).
    • Protein or peptide: Tubulin beta chain
    • Protein or peptide: Tubulin alpha chain
  • Ligand: GUANOSINE-5'-DIPHOSPHATE
  • Ligand: GUANOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Undecorated tubulin lattice following polymerisation with GTP (no...

SupramoleculeName: Undecorated tubulin lattice following polymerisation with GTP (no stabilising ligands).
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Details: The native tubulin was isolated from the Xenopus eggs using TOG-affinity chromatography. Microtubules were polymerised on the cryo-EM grid.
Source (natural)Organism: Xenopus tropicalis (tropical clawed frog) / Tissue: Oocyte / Location in cell: cytoplasm

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Macromolecule #1: Tubulin beta chain

MacromoleculeName: Tubulin beta chain / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Xenopus tropicalis (tropical clawed frog) / Tissue: oocyte
Molecular weightTheoretical: 49.87877 KDa
SequenceString: MREIVHLQAG QCGNQIGAKF WEVISDEHGI DPTGTYHGDS DLQLERINVY YNEATGGKYV PRAVLVDLEP GTMDSVRSGP FGQIFRPDN FVFGQSGAGN NWAKGHYTEG AELVDSVLDV VRKEAESCDC LQGFQLTHSL GGGTGSGMGT LLISKIREEY P DRIMNTFS ...String:
MREIVHLQAG QCGNQIGAKF WEVISDEHGI DPTGTYHGDS DLQLERINVY YNEATGGKYV PRAVLVDLEP GTMDSVRSGP FGQIFRPDN FVFGQSGAGN NWAKGHYTEG AELVDSVLDV VRKEAESCDC LQGFQLTHSL GGGTGSGMGT LLISKIREEY P DRIMNTFS VVPSPKVSDT VVEPYNATLS VHQLVENTDE TYCIDNEALY DICFRTLKLT TPTYGDLNHL VSATMSGVTT CL RFPGQLN ADLRKLAVNM VPFPRLHFFM PGFAPLTSRG SQQYRALTVP ELTQQMFDAK NMMAACDPRH GRYLTVAAVF RGR MSMKEV DEQMLNVQNK NSSYFVEWIP NNVKTAVCDI PPRGLKMSAT FIGNSTAIQE LFKRISEQFT AMFRRKAFLH WYTG EGMDE MEFTEAESNM NDLVSEYQQY QDATAEEEGE FEEGEEEENA

UniProtKB: Tubulin beta chain

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Macromolecule #2: Tubulin alpha chain

MacromoleculeName: Tubulin alpha chain / type: protein_or_peptide / ID: 2 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Xenopus tropicalis (tropical clawed frog) / Tissue: Oocyte
Molecular weightTheoretical: 50.114223 KDa
SequenceString: MRECISIHIG QAGVQMGNAC WELYCLEHGI QQDGIIPDEK TAATDSSFGT FFSETGSGKH VPRAVFVDLE QTVIGEIRTG HYRSLFHPE QLITGKEDAA NNYARGHYTI GKEIVDSVLD RVRKMADQCS GLQGFLIFHS FGGGTGSGFT SLLMERLSVD Y GKKSKLEF ...String:
MRECISIHIG QAGVQMGNAC WELYCLEHGI QQDGIIPDEK TAATDSSFGT FFSETGSGKH VPRAVFVDLE QTVIGEIRTG HYRSLFHPE QLITGKEDAA NNYARGHYTI GKEIVDSVLD RVRKMADQCS GLQGFLIFHS FGGGTGSGFT SLLMERLSVD Y GKKSKLEF SVYPAPQIST AVVEPYNSIL TTHTTLEHSD CAFMVDNEAI YDICNRNLDI ERPTYTNLNR LIGQIVSSIT AS LRFDGAL NVDLTEFQTN LVPYPRIHFP LVTYSPIISA EKAYHEQLSV PEITNACFEY SNQMVKCDPR RGKYMACCLL YRG DVVPKD VNAAIAAIKT RRSIQFVDWC PTGFKVGINY QPPTVVPGGD LAKVQRAVCM LSNTTAIAEA WARLDHKFDL MYSK RAFVH WYVGEGMEEG EFSEAREDMA ALEKDYEEVG TESGDGGDEE EDEY

UniProtKB: Tubulin alpha chain

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Macromolecule #3: GUANOSINE-5'-DIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-DIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 6 / Formula: GDP
Molecular weightTheoretical: 443.201 Da
Chemical component information

ChemComp-GDP:
GUANOSINE-5'-DIPHOSPHATE / GDP, energy-carrying molecule*YM

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Macromolecule #4: GUANOSINE-5'-TRIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 6 / Formula: GTP
Molecular weightTheoretical: 523.18 Da
Chemical component information

ChemComp-GTP:
GUANOSINE-5'-TRIPHOSPHATE / GTP, energy-carrying molecule*YM

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 6 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation statefilament

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Sample preparation

Concentration1.6 mg/mL
BufferpH: 6.8
Component:
ConcentrationNameFormula
80.0 mMPIPES
1.0 mMEGTA
1.0 mMMagnesium chlorideMgCl2
1.0 mMGuanosine triphosphateGTP

Details: BRB80 + 1 mM GTP
GridModel: C-flat-2/2 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 40 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.00025 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 303 K / Instrument: LEICA EM GP
Details: The sample was incubated on the grid for 10 minutes within the Leica chamber to allow for tubulin polymerisation..

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 6200 / Average exposure time: 3.4 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.1 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 81000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 192000
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Software - details: AutoRefine
Details: These are pseudo-particles following the symmetry expansion of ~46,000 particles
Number images used: 486937
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: Other / Chain - Initial model type: other
Details: 14-3 X. tropicalis tubulin dimer from this publication.
DetailsInitially a tubulin dimer was docked into the density within ChimeraX, then molecular dynamics force field-based model fitting was carried out by ISOLDE using unsharpened and deepEMhancer processed density. The model was refined through iterations of ISOLDE and Phenix real_space_refinement softwares.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-9g0t:
Xenopus tropicalis undecorated microtubule - 15 protofilament, 3-start helix

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